The shikimate pathway is an ancient pathway that is involved in primary and secondary metabolism and is found in all prokaryotes, many lower eukaryotes, and plants, but not in mammals. In primary metabolism, the function of the pathway is to provide the precursors for the production of the aromatic amino acids and para-aminobenzoic acid. The shikimate pathway includes the enzymes and metabolites formed by converting 3-Deoxy-D-arabino-heptulosonic 3-phosphate (DAHP) to chorismic acid, the trifurication point for the three pathways leading to the production of tryptophane, tyrosine, and phenylalanine.
The importance of the shikimate pathway to cell viability is illustrated by experiments that result in the disruption of enzyme function. In plants, the shikimate pathway enzyme, EPSP synthase, has been targeted by a chemical inhibitor strategy that has resulted in the commercially successful, broad range, post-emergent herbicide called glyphosate. Glyphosate inhibits the shikimic acid pathway, which leads to the biosynthesis of aromatic compounds including amino acids, plant hormones, and vitamins. Specifically, glyphosate inhibits the conversion of phosphoenolpyruvic acid (PEP) and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acid by binding to the enzyme 5-enolpyruvyishikimate-3-phosphate synthase (hereinafter referred to as EPSP synthase or EPSPS).
In various microbial species, analysis of the shikimate pathway has been carried out genetically by the construction of mutants. When mutants of virulent prokaryotic or microbial eukaryotic species lacking enzymes at various steps in this pathway, the so-called aro− mutants, are used to infect animals, their virulence is generally observed to be attenuated (Leech et al. 1995 J. Biol. Chem. 270:25827-25836 and Gunel-Ozcan et al. 1997. Microbial Pathogen 17:169-174). After infection with aro− mutants of S. typhimurium, mice are resistant to further challenge with the wild type strain.
Recently, the shikimate pathway has been characterized in apicomplexan parasites such as Toxoplasma gondii, Plasmodium falciparum (malaria) and Cryptosporidium parvum (Roberts et al. 1998. Nature 393:801-805). In addition, Roberts et al. reported that the growth of these parasites can be inhibited by glyphosate.
The observations that both chemical and genetic inhibition of the shikimate pathway results in reduced cell viability has stimulated interest in the pathway as a possible target for drug therapy in acute microbial infection. It is likely that compounds which can inhibit the activity of shikimate enzymes will not cause cell death of the infecting microbe, but will result in attenuation in a manner analagous to the phenotype of shikimate pathway mutants. As antimicrobials, these compounds may be expected to induce stasis rather than cell lysis or death, allowing the infection to be cleared by the host's immune system. Such an outcome is desirable as it will ameliorate the absolute selective pressure to select for the growth of resistant mutants which would inevitably be the case if the compounds used caused cell death. Additionally this strategy may also result in a degree of immune protection which may prevent reinfection. As efficacious compounds are unlikely to kill any infecting microorganisms, then the risks of toxic shock caused by, for example, bacterial protein and cellular debris will be minimized when treatment is administered.
Protozoan parasites of the phylum Apicomplexa include the causative agents of the human disease malaria, as well as the agents of cattle diseases such as Texas cattle fever and East Coast fever. Furthermore, the causative agent of the human disease toxoplasmosis, Toxoplasma gondii, is also found in this phylum (Schmidt, G. D. and Roberts, L. S. 1985. Foundations of Parasitology. St. Louis, Times Mirror/Mosby, pp. 149, 173-178).
Malaria is one of the most important diseases of mankind. Two billion people are at risk of contracting malaria; over 200 million people are infected by the disease, and 3 million people die of malarial infection each year. The disease is caused by four species of plasmodia, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. Strains of the most common and most severe causative agent, P. falciparum, have developed resistance to many of the current drugs used in treatment, and drug resistance has also been reported in P. vivax (Pudney, M. “Antimalarial: From Quinine to Atovaquone” in: Hunter, P. A., Darby, G. K. and Russell, N. J. Fifty Years of Antimicrobial: Past Perspectives and Future Trends (Cambridge, Society for General Microbiology, 53rd Symposium, 1995), pp. 229-247).
Chemical agents belonging to the triazine class of herbicides have been suggested as potential therapeutic agents. Such activity against some apicomplexan parasites is thought to result from interaction of the herbicide with the D1 protein of the photosynthetic reaction center of organelles of the parasites (Hackstein, J. H. P. et al. 1995. Parasitology Research 81:207-216). In addition, dinitroaniline herbicides known to be inhibitors of plant microtubules also inhibit some apicomplexan parasites (Stokkermans, T. J. W. et al. 1996. Experimental Parasitology 84:355-370). Others have nominated herbicidal agents which inhibit carotenoid synthesis or certain herbicidal agents which inhibit fatty acid synthesis as inhibitors of apicomplexan parasites (see U.S. Pat. No. 5,877,186).
Recently, oxalic acid, a dicarboxylic acid, was shown to enhance the herbicidal efficacy of glyphosate (See U.S. Pat. No. 5,863,863). The mode of action of oxalic acid, however, was attributed to its ability to interact with cationic amine surfactants and oxalic acid was formulated in the form of an enhancer composition containing oxalic acid and cationic surfactants, which was then used to dilute commercial glyphosate formulations. Oxalic acid and other polyvalent anions that are good chelators have been shown to enhance glyphosate performance by sequestering bivalent cations. However, use of complexing agents as additives to glyphosate has been reported in the literature.